1. Field of the Invention
The present invention relates to cell culture. In particular, this invention is directed to methods and apparatuses in two or three-dimensional architecture used to observe or quantitate cell proliferation in the presence of potential growth promoting molecules.
2. Description of Relevant Art
Tissue engineering is based on the concept that new tissues such as skin or bone may be formed by starting with building materials (e.g., natural polymers, such as collagen, synthetic polymers and/or inorganic composites) and then shaping the materials into a three-dimensional scaffold. The scaffold is seeded with living cells and exposed to growth factors. When the cells multiply, they fill up the scaffold, grow into three-dimensional tissue, and recreate their intended tissue functions once implanted into the body. Blood vessels attach themselves to the new tissue, the scaffold dissolves, and the newly-grown tissue eventually blends in with its surroundings.
In contrast to conventional 2-dimensional cell culture systems, e.g. culture dishes or multi-well tissue culture plates, scaffolds mimic the complex 3-dimensional cellular structure of living tissues by providing not only an adhesive substrate for cells, but by acting as a 3-dimensional physical support for in vitro culture and, in some cases, for subsequent implantation.
Cell and tissue function may be dependent on scaffold morphology and the materials used to make the scaffold. A large surface area to volume ratio within 3D structures is necessary to support the adhesion of a large number of cells. Porosity also needs to be adequate to provide enough space to allow a cell suspension to penetrate the 3D structure. Additionally, texture, roughness, hydrophobicity, charge and chemical composition are surface properties known to affect cell adhesion and subsequent cell behavior on a polymer surface.
Extracellular matrix (ECM) molecules may act to enhance these surface properties. ECM molecules consist of secreted proteins and polysaccharides which can be derived from some tissues of multicellular organisms. The ECM occupies the intercellular space and binds cells and tissues together. Cells can attach to matrix proteins by interacting with them through cell adhesion molecules such as integrins.
ECM molecules may act to enable cell proliferation or differentiation. For example, a scaffold comprised of polyhydroxyethylmethacrylate did not result in cultured nerve cells showing nerve growth unless fibronectin, an extracellular matrix protein, was incorporated into the scaffold Scaffolds comprised of polylactic acid and collagen, another ECM protein, resulted in the proliferation of bovine articular chondrocytes whereas scaffolds comprised of some other materials did not result in such proliferation.
Specific ECM proteins may enable cell growth and/or differentiation alone or in conjunction with growth factors. Morphological changes induced by recombinant growth and differentiation 5 factor (GDF-5) in fetal rat calvarial cells marked by cellular aggregation and nodule formation is dramatically synergized by the presence of Type I collagen, but not fibronectin. Moreover, this synergistic effect is highly specific to GDF-5 as compared to other mitogens which failed to induce a similar response. This finding highlights the importance of identifying optimal combinations of extrinsic factors required for growth of cells in vitro and the necessity of designing scaffolds with appropriate materials.
The basis for assaying the different properties of scaffolds, the materials which comprise them, and associated bioactive agents on cell function, is the need to count cells. Counting cells, however, may be a time consuming process with two dimensional cell cultures and difficult with three-dimensional cultures. Approaches include releasing cells from a surface by trypsin and then counting them directly by Coulter Counter or hemacytometer. Problems with counting procedures arise because real time measurements cannot be taken for two or three-dimensional cultures.
Additionally, taking this approach with three-dimensional scaffolds is even more problematic. Since scaffolds are porous and three-dimensional, there are problems of diffusion within the material and the trypsin may not be able to reach all of the cells in the interior regions of the scaffold.
Another option would be to perform a metabolic assay such as the MTT assay where the cells' reduction of the tetrazolium salt 3,[4,5-Dimethylthiazol-2-yl]-2-5 diphenyltetrazolium bromide (MTT) is measured. Likewise, for three-dimensional culture, the MTT may not be able to reach all the cells in a scaffold. Other disadvantages with this method include the multiple reagent additions which are required and the fact that the test itself is non-reversible. Further time point readings of the same cell cultures cannot be performed without setting up a separate assay to be used for each time point.
A third approach for cell counting is to quantitate the contents (usually DNA or protein) of the cells and compare them to a standard curve. In a DNA assay, a dye will bind to the DNA of lysed cells and exhibit strong fluorescence. However, DNA assays may result in the DNA sticking to the scaffold material and, therefore, decreasing the actual level of fluorescence in a cell solution resulting in an inaccurate count. This method also requires setting up separate assays for each time point.
Cell counting may also be accomplished using, for example, a BD Oxygen Biosensor (Becton Dickinson, Bedford, Mass.). Unlike other methods, the fluorescent BD Oxygen Biosensor assay does allow for real time noninvasive monitoring of cellular growth. The assay is based upon the measurement of oxygen dissolved in assay mediums. The BD Oxygen Biosensor uses the fluorescence of ruthenium dye that is quenched in the presence of oxygen. The dye is immobilized within an inert but highly oxygen-permeable silicone matrix. Previous data suggest that increase in cell number correlates well with an increase in oxygen consumption.
Although this apparatus is known to work well with some cell types, adherent cells may be difficult to grow in certain biosensor plates and may generate a large fluorescent signal when they do grow. The silicone surface of the sensor does not support growth of many cell types and provides a small surface area. Contact-inhibited cells may not grow in large enough numbers to generate a sufficient oxygen sink to change the sensor fluorescence.
Clearly, there is a need for devices which will easily enable researchers to test different molecules, such as ECMs or other materials used or incorporated into three dimensional scaffolds, for their effects on cell proliferation. There is also a need for methods or devices which enable the simple assaying of cell proliferation in three dimensional cell scaffolds or three dimensional cultures in real time.